Spindle pole body (SPB) duplication during the budding yeast cell cycle is a critical and essential event in assembly of the mitotic spindle that ultimately segregates the chromosomes to the progeny cells. In yeast, the SPB is the sole microtubule organizing center and is embedded in the nuclear envelope (NE) nucleating microtubules in the nucleoplasm and in the cytoplasm. Failure in SPB duplication or in SPB function leads to anueploidy and polyploidy similar to that observed in many cancer cells. This proposal addresses the function of two genes, NDC1 and MPS2, that are required during SPB duplication to insert the new SPB into the NE. We have found that these essential genes encode membrane proteins that localize to the SPB. In addition, Ndc1p is found in nuclear pore complexes (NPC) and Mps2p is found in the NE and the ER membranes. NPCs, like SPBs, are embedded in the NE.
The aims of this proposal are to first, explore the function of Ndc1p and Mps2p in SPB duplication by looking for genetic and physical interactions with other SPB components. We will also carry out genetic screens to look for additional genes that function in SPB duplication. Second, we will determine the topology of Ndc1p and Mps2p in the nuclear envelope. Furthermore, we will determine whether these proteins have access to the nucleoplasm and/or the cytoplasm as part of our efforts to determine how they function. Third, we will study the NPC-related function of Ndc1p. We believe that Ndc1p may be required for NPC assembly, like SPB assembly. We propose a variety of genetic, cytological and biochemical approaches to discover the role of Ndc1p at NPCs and to identify proteins with which it collaborates in this function. Finally, we have identified a novel gene, NOE1, that is required for the viability of ndc1-1 cells and whose product may be localized to the SPB, suggesting a possible role for Noe1p in SPB duplication. We will define the role of Noe1p with respect to Ndc1p, and identify genes that interact with NOE1. The proposed project is aimed at understanding the function of a set of unusual membrane proteins at the SPB and the NPC, and at identifying other proteins with which the interact at these organelles.
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